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The Distribution of Cytochrome C in Developing Pollen of Normal and S
Lindsey Verduin and Christine D. Chase
Apoptosis, or programmed cell death, is a known regulatory feature of eukaryotic cells. In mammalian
cells, apoptosis is often initiated by the release of cytochrome c from the mitochondria. However, it is
unknown whether cytochrome c plays a role in cell death signaling in plants. We hypothesized cytochrome c
released from the mitochondria causes pollen collapse in S male-sterile maize. Mitochondrial pellet and
post-mitochondrial supernatant samples were collected from developing sterile and normal pollen. The location
and relative abundance of cytochrome c in these samples was detected by Western blot analysis. A
large concentration of cytosolic cytochrome c was detected in both sterile and normal samples, contradictory to
the expected lack of the protein in normal cytosolic fractions. This finding indicates that cytochrome c release
from mitochondria does not, by itself, initiate cell death.
Apoptosis, or programmed cell death, is a mechanism found in vertebrates allowing individual cells to die
and disintegrate in a regulated process. While apoptosis is often associated with disease, it is also a
necessary cellular function, involved in the immune response and the destruction of worn out cells1. Cell death can
be induced by a number of factors but the main executers in all apoptotic pathways are caspases. Caspases are
a family of proteases that, once activated, initiate a caspase cascade, acting on various cellular substrates,
to culminate in cell death2.
Various signals and organelles can induce apoptosis, but perhaps the most well known cell death pathway
involves the mitochondria. Proapoptotic proteins, such as Bax and Bid of the Bcl-2 family, interact with the
outer mitochondrial membrane, forming openings in the mitochondria3. This leads to mitochondrial
depolarization, swelling, and ultimately, the release of its proteins3. The most significant protein released
is cytochrome c, which is part of the oxidative phosphorylation pathway. Upon its release, cytochrome binds
with Apaf-1, forming an apoptosome2. This complex activates caspase 9, initiating the "death cascade"2.
While apoptosis is a common feature in mammalian cells, it is unknown whether plants, specifically maize, follow
the same apoptotic cell death pathway. As in animals, plant cell death is a common regulatory process, important
in a plant's sexual maturation and the growth of numerous structures4. Insight into this issue could be found
with studies on maize cytoplasmic male sterility (CMS). Cytoplasmic male sterility is an occurrence in many
plants that results in the production of nonfunctional pollen5. The cause of pollen collapse has not yet
been determined. However, the expression of two mitochondrial reading frames unique to CMS-S maize, orf355
and orf77, may be responsible for pollen collapse in this system5. The exact mechanism of orf355 and orf77
sterility is unknown. Transcription and translation of orf77 can result in a truncated protein called orfl75. Orfl7
has homology with the mitochondrial ATP synthase protein ATP9, specifically in the transmembrane domain. It
is thought that orf355 and orf77 may disrupt the assembly of ATP synthase leading to pore formation in
the mitochondrial membrane and the subsequent release of mitochondrial proteins5.
The purpose of this research was to determine the location and relative abundance of cytochrome c in CMS-S
maize cells. From this, it could be possible to ascertain whether cytochrome c plays a role in cell death. A
study conducted by Lee et al. pertaining to pollen abortion in CMS maize described mitochondrial swelling prior
to pollen cell death, which is similar in morphology to mitochondria in mammalian cells undergoing apoptosis6.
Based on this morphological similarity and Western blot results showing collapsed pollen mitochondria depleted
of cytochrome c, we hypothesized that cytochrome c could be a cell death signal in plants. Our hypothesis
predicts that if cytochrome c acts a cell death signal, the majority of cytochrome c should be located in the cytosol
of collapsed pollen, while normal pollen granules should have cytochrome c concentrated within the mitochondria.
Fig. 1 shows the procedure for a cell fractionation. Zea mays (corn) pollen was obtained from Mo17N and
Mo17S lines, normal and sterile respectively. Pollen was recovered from each line at various stages of the corn's
life cycle, including normal starch filling pollen, sterile collapsed pollen, and normal and sterile microspores.
The microspore is an early stage in pollen development. Mo17S and Mo17N microspores are
morphologically indistinguishable. Mo17S pollen collapses abruptly later in pollen development.
A pollen sample (1-2g) was crushed in mitochondrial grinding buffer (0.5M sucrose, 50mM Trizma base, imM
EGTA, 10mM KH2PO4, pH 7.6; BSA, 2-mercaptoethanol, 0.1M PMSF) using a mortar and pestle. Following
the protocol, two filtrations were performed to isolate cellular components. The filtrate was centrifuged three
times, with the centrifugations being performed on each successive supernatant collected. The end product was
a mitochondrial pellet sample and a post-mitochondrial supernatant sample. 10% of each mitochondrial pellet
and supernatant fraction was examined to determine relative cytochrome c abundance among the samples.
300 ul crude exht
+ 75 l 6X NUPAGE $8
70oC 10 min
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Recove sup for g hez:
38 ul = 1% staring marnal
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Recover sup for gel; free;
38 Lu= 1% Wtarting meal
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Reower sup for gel; freeze;
38 ul= 1% starting male
3000 ul aude etrat
2,700 ul crude extact
(2 x 1,350 li)
1500 x g, 4oC 15min
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15,001 xg.4oC, 15 min
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Prep for BNGEe
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IS 00 g Wcletfs = nitodthrdriar
+ 150 ul iI NUPAGE 58 e3th
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Rewr sup 'of gel ftrze 3 8 I a 1%
Figure 1. Cell fractionation diagram
The protein samples obtained from cell fractionations were separated on 12% NuPAGEï¿½ Bis-Tris gel
(Invitrogen; Carlsbad, CA). Varying amounts of protein samples (3.8 - 25 ul) were mixed with lx sample buffer
(5x sample buffer, DTT, 0.1% bromphenol blue, 0.1% PMSF) and loaded on the gel. Molecular weight
markers (SeeBlue5 Plus2 Pre-Stained Standard) were loaded in lane 1 of every gel. The gel was run in lx
NuPAGEï¿½ MES SDS Running Buffer, with NuPAGEï¿½ antioxidant added to the inner chamber, according
to manufacturer's instructions (XCell Sure LockTM Mini-Cell). Electrophoresis was carried out at 100V for about
2 hours, or until dye marker reached 2 cm from the bottom of the case.
Proteins from the gel were transferred to a nitrocellulose membrane (Invitrogen 0.2 pm pore nitrocellulose/
filter paper sandwich; XCell IITM Blot Module). The blot was incubated in blocking solution (2% or 3% w/v BSA
in TBST) for 1 hour, washed in TBST (NaCI 0.14M; KCI 2.7mM; 1M Tris, pH8), and incubated 1 hour in
primary antibody. Cytochrome c monoclonal primary antibody was obtained from PharMingen (clone
7H8.2C12). ATPy was an affinity purified polyclonal primary antibody. Cytochrome c and ATPy were both diluted
at 10pl/50ml TBST. The blot was washed 6 times in TBST in 10 min intervals, followed by incubation for 1 hour in
a horseradish peroxidase linked secondary antibody (mouse or rabbit at lpl/40ml TBST). The blot was again
washed 6 times in TBST. Reactivity was observed using chemiluminescence (SuperSignal, Pierce)
and autoradiographic film.
Figure 2.1 shows an anti-cytochrome c blot. Comparison with the molecular weight markers showed a 14
kDa protein, which was in keeping with the known weight of cytochrome c (12 kDa). The relative abundance
of cytochrome c in each lane was compared between sterile and normal samples at distinct life stages. Sterile
and normal immature ear samples were included to determine if cytochrome c release from the mitochondria
could be a developmental feature in the maize life cycle. The sterile and normal immature ear and
microspore samples showed no difference in cytochrome c abundance in either the pellet or the supernatant.
An unexpected result was the observation of large amounts of cytosolic cytochrome c in all the samples.
However, sterile collapsed pollen does appear to have the highest abundance of cytochrome c in the cytosol.
Figure 2.2 shows an anti- ATPy blot. ATPy is a subunit of ATP synthase, which is a mitochondrial inner
membrane structure. The ATPy blot was performed on the blot from Fig. 2.1 and included as a control to
assess membrane integrity. The predominance of ATPy in the pellet samples shows that the mitochondria are
largely intact. The BSA present in supernatant fractions caused artifactual lane narrowing for the
2 3 4 5 6 7 8 9 10 11
Figure 2.1. Results of Western blot against cytochrome c. Lane 2,3: sterile immature ear
mitochondrial pellet (P) and post-mitochondrial supernatant (S). Lane 4,5: normal immature ear P and
S. Lane 6,7: sterile microspore (msp) P and S. Lane 8,9: normal msp P and S. Lane 10,11:
sterile collapsed pollen P and S. All samples were prepared in 0.5M sucrose and 1% protease-free BSA.
Figure 2.2. Results of Western blot against ATPy. Lane samples correspond to Fig. 2.1.
Figure 3 shows results from an anti-cytochrome c blot. The samples in Fig. 3 were prepared in delipidated BSA,
as opposed to 1% protease-free BSA. Delipidated BSA is known to protect membrane integrity, prompting us
to compare with protease-free BSA to determine if the delipidated BSA affected cytochrome c release. The
results from Fig. 3 are identical to Fig. 2.1, indicating that the source of BSA has no effect on cytochrome c
release. Fig. 3 shows more clearly the unexpectedly high amount of cytosolic cytochrome c in the normal
microspore and starch filling pollen. However, there is a striking difference evident between sterile collapsed
pollen and normal starch filling pollen cytochrome c abundance. The majority of cytochrome c in collapsed pollen
is released into the cytosol, while starch filling pollen has an equal proportion of cytochrome c in its mitochondria
According to a study by Petrussa et al., when mitochondria are suspended in a medium containing potassium,
the mitochondrial K+ATP channel is opened, causing the organelle to swell and release cytochrome c7. Based on
this fact, it was thought that potassium from the KH2PO4 found in the mitochondrial grinding buffer might have
been affecting the amount of cytochrome c released from the mitochondria. Lanes 11 and 12 of Figure 3 show
sterile microspore samples prepared in grinding buffer containing no KH2PO4. The absence of potassium had no
effect on cytochrome c abundance, as can be seen by comparing lanes 11 and 12 with lanes 2 and 3.
2 3 4 5 6 7 8 9 10 1112
Figure 3. Results of Western blot against cytochrome c. Lanes 2,3: sterile msp P and S. Lanes 4,5:
normal msp P and S. Lanes 7,8: sterile collapsed pollen P and S. Lanes 9,10: normal starch filling pollen
P and S. Lanes 11,12: sterile msp P and S prepared in potassium free grinding buffer. All samples
were prepared in 0.5M sucrose and delipidated BSA.
The abundance of cytochrome c in the supernatant of the normal starch filling pollen was unexpected. The
sucrose concentration in the mitochondrial grinding buffer was altered to assess whether it played any role
in cytochrome c release. Figure 4 shows an anti-cytochrome c blot on sterile and normal microspores prepared
in 0.065M and 0.8M sucrose concentration. Increasing the sucrose concentration from 0.5M did not have any
effect on the amount of cytochrome c released in the samples.
2 3 4 5 6 7 8 9 10 11
Figure 4. Results of Western blot against cytochrome c. Lanes 2,4: sterile msp P and S in 0.65M
sucrose. Lanes 6,8: normal msp P and S in 0.65M sucrose. Lanes 10,11: normal msp P and S in
0.8M sucrose. All samples were prepared using delipidated BSA.
The hypothesis that cytochrome c is a plant cell death signal was not fully demonstrated. While sterile
collapsed pollen mitochondria were found to be largely void of cytochrome c, this does not conclusively indicate
that cytochrome c is causing the pollen collapse. The finding that both collapsed pollen and normal starch
filling pollen have large amounts of cytochrome c in the cytosol indicate that cytochrome c may not play a role in
cell death signaling in plants. This theory is supported in a recent study by Yao et al. which showed that
while cytochrome c is released during plant cell death, cytochrome c itself is not sufficient to induce apoptosis8.
The finding that sterile and normal microspores showed the same cytochrome c abundance pattern (a large
amount present in the cytosol) was unexpected. As mentioned above, the presence of a large cytosolic cytochrome
c concentration in normal starch filling pollen was surprising. These results could have been caused by various
factors not taken into previous consideration. The three experiments performed to account for these
discrepancies, removing potassium from the grinding buffer, increasing sucrose concentrations, and substituting
1% protease BSA, had no effect on cytochrome c concentrations.
One aspect that may be relevant to future experimentation is the form of cytochrome c that is present in the
cytosolic samples. Newly synthesized cytochrome c present in the cytoplasm is in the "apo" form. The "apo"
form contains no heme group and is not functional until it is transported into the mitochondria. Once inside
the mitochondria, a heme group is added and cytochrome c is converted to the "holo" form9.
The hypothesis predicts that the cytochrome c in microspore supernatants would be in the "apo" form,
while collapsed pollen supernatant should be mainly "holo" cytochrome c. A series of experiments utilizing the
ability of heme to direct chemiluminescence was conducted. The above mentioned protocol of fractionation
and electroblotting was followed to produce new cellular samples. SuperSignal (Pierce) luminol was added directly
to the nitrocellulose membrane in the absence of antibodies10. However, the chemiluminescent reaction was
not sensitive enough to react under our extraction conditions. This topic of study will pave the way for a new line
of experimentation that is capable of detecting and distinguishing the "apo" and "holo" form of cytochrome c.
Future experiments will be performed involving laser confocal microscopy to pinpoint where exactly the cytochrome
c is located within the cell9.
Discovering the intricate pathways of plant cell death has important agricultural and economic implications.
By understanding how plant death occurs, it may be possible to prevent or induce cell death, depending upon
the situation. Knowledge concerning plant cell death is a gray area but full of numerous avenues of study to
unravel its causes and pathways.
1. Lemasters JJ. Dying a thousand deaths: redundant pathways from different organelles to apoptosis and
necrosis. Gastroenterology 2005;129:351-60.
2. Lavrik IN, Golks A, Krammer PH. Caspases: pharmacological manipulation of cell death. Journal of Clinical
3. Green GR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626-29.
4. van Doorn WG, Woltering EJ. Many ways to exit? Cell death categories in plants. Trends in Plant
5. Gallagher LJ, Betz SK, Chase CD. Mitochondrial RNA editing truncates a chimeric open reading frame associated
with S male-sterility in maize. Current Genetics 2002;42:179-84.
6. Lee SJ, Earle ED, Gracen VE. The cytology of pollen abortion in S cytoplasmic male-sterile corn anthers.
American Journal of Botany 1980;67:237-45.
7. Petrussa E, Casolo V, Peresson C, Braidot E, Vianello A, Marci F. The K+ATP channel is involved in a low-
amplitude permeability transition in plant mitochondria. Mitochondrion 2004;3:297-307.
8. Yao N, Eisfelder BJ, Marvin J, Greenberg JT. The mitochondrion- an organelle commonly involved in programmed
cell death in IArabidopsis thaliana. The Plant Journal 2004;4:596-610.
9. Oliver L, LeCabellec MT, Pradal G, Meflah K, Kroemer G, Vallette FM. Constitutive presence of cytochrome c in
the cytosol of a chemoresistant leukemic cell line. Apoptosis 2005;10:277-87.
10. Vargas C, McEwan AG, Downie JA. Detection of c-type cytochromes using enhanced chemiluminescence.
Analytical Biochemistry 1993;209:219-23.
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